Triple combination formulation for treatment of chronic pain

ABSTRACT

The disclosure relates to use of a triple formulation comprising a histone deacytlase, a cyclodextrin and polyethylene glycol or propylene glycol, and in the treatment and management of chronic pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/209,261, filedDec. 4, 2018, which claims priority to U.S. Provisional Application No.62/594,216, filed Dec. 4, 2017, both of which are incorporated in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DE022746 andNS035115 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

The field of the invention is related to therapeutics and methods oftreating chronic pain.

Chronic pain affects at least 100 million American adults—more than thetotal affected by heart disease, cancer, and diabetes combined. Chronicpain remains the number one source of disability in the US and 5thworldwide. Pain also costs the nation around $500 billion each year inmedical treatment and lost productivity. Every new epidemiological studyindicates that these rates continue to increase worldwide. Treatmentoptions for chronic pain remain limited. The majority of chronic painpatients are not satisfied with their pain management, as pain relief isshort in duration, associated with adverse effects, and the mosteffective options, namely opioids, are linked to addiction and relatedmortalitys at an epidemic rate⁶. Thus, novel, non-opioidergic treatmentoptions are urgently needed for chronic and especially for neuropathicpain conditions.

There is a need for new therapeutic formulations for treatment ofchronic pain.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned drawbacks byproviding formulations and methods of treating chronic pain.

In one aspect, the disclosure provides triple combination formulationsfor the use in the treatment and management of chronic pain.

In one aspect, the disclosure provides a method of treating chronic painin a subject in need thereof, the method comprising administering to thesubject a composition comprising (i) a histone deacetylase (HDAC)inhibitor, (ii) a cyclodextrin or salt thereof, and (iii) polyethyleneglycol (PEG) or propylene glycol, wherein the composition is provided ina therapeutically effective amount to treat the chronic pain. In oneaspect, the HDAC inhibitor is vorinostat. In one aspect, thecyclodextrin is 2-hydroxypropyl-b-cyclodextrin (HPBCD). In one aspect,the polyethylene glycol (PEG) or propylene glycol is polyethyleneglycol. In one further aspect, the composition comprises, consists of orconsists essentially of vorinostat as the HDACi,2-hydroxypropyl-b-cyclodextrin (HPBCD) as the cyclodextrin, andpolyethylene glycol.

In another aspect, the present disclosure provides a method of reducingor inhibiting one or more symptoms of chronic pain, the methodcomprising administering to the subject a composition comprising (i) ahistone deacetylase (HDAC) inhibitor, (ii) a cyclodextrin or saltthereof, and (iii) polyethylene glycol (PEG) or propylene glycol,wherein the composition is provided in a therapeutically effectiveamount to reduce or inhibit one or more symptom of chronic pain. In oneaspect, the HDAC inhibitor is vorinostat. In one aspect, thecyclodextrin is 2-hydroxypropyl-b-cyclodextrin (HPBCD). In one aspect,the polyethylene glycol (PEG) or propylene glycol is polyethyleneglycol. In one further aspect, the composition comprises, consists of orconsists essentially of vorinostat as the HDACi,2-hydroxypropyl-b-cyclodextrin (HPBCD) as the cyclodextrin, andpolyethylene glycol.

In another aspect, the disclosure provides a use of a compositioncomprising (i) a histone deacetylase (HDAC) inhibitor, (ii) acyclodextrin or salt thereof, and (iii) polyethylene glycol (PEG) orpropylene glycol for manufacture of a medicament for the treatment ofchronic pain or inhibition of symptoms of chronic pain.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere are shown, by way of illustration, preferred embodiments of theinvention. Such embodiments do not necessarily represent the full scopeof the invention, however, and reference is made therefore to the claimsand herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts the working model of action for the triple combinationformulation (TCF), adapted from US 2015/0359762, incorporated byreference in its entirety.

FIGS. 2A-2B depict the increased plasma and brain concentration ofvorinostat (Vo) in TCF injected mice. A. Plasma. Npc1^(+/nmf164)injected i.p. with Vo or TCF. Blood at 1h post injection. Mean±SEM fromtwo independent experiments shown (5 mice/group, each experiment).*p<0.05, TCF vs Vo. B. Pharmacokinetics of Vo in mouse brain.Npc1^(+/nmf164) mice injected i.p. with Vo or TCF. At indicated times,animals sacrificed, perfused with PBS and brain was harvested. n=10 miceat 0.5 h; n=5 remaining time points. *p<0.05. Vo conc. determined bymass spec. Alam et al.¹

FIGS. 3A-3B show acetylation of histone 3 and 4 in brain ofNpc1^(nmf164) mice. A. Western blots show acetylation of histones 3 and4 (H3 and H4) in brain of Npc1^(nmf164) mice at 60 min afteradministration of TCF, its components or vehicle control. n=3 (3 mice ineach n). Coomassie stained gel (CBB; blue) confirmed equal sampleloading. B. Quantitation of A. Fold change relative to Vo (set at 1).*p<0.05. Alam et al.¹

FIG. 4 depicts indirect fluorescence antibody (IFA) detection ofmicroglial cells (green) with anti-Iba1 antibodies in hippocampus ofmice untreated (Un) and TCF treated at 8 months. Schematic shows areaBlue, DAPI. Scale bar, 40 μm. Per group, n=2 mice. Two sagittal brainsections from each mouse were examined.

FIGS. 5A-5B depict no loss of Purkinje neurons in the cerebellum orinflammation in the hippocampus for mice treated. A. Quantitativeanalysis of Iba1 positive microglial cells in the hippocampus, shown inFIG. 4. B. Purkinje cell counts from representative images of IX lobuleof the cerebellum from untreated (Un) and TCF treated Balb/c mice at 8months. For quantitative analyses of TCF treatment, 4 mice were utilizedto yield 16 sections (4 per mouse). In untreated animals, 2 mice wereutilized to yield 8 sections (4 per mouse).

FIGS. 6A-6D depict analgesia, body weight, and mobility of spared nerveinjury (SNI) rats treated with TCF or vehicle, monitored over 1 year. A.Tactile sensitivity (50% threshold) monitored for SNI-injured paw,labeled as (SNI), and contralateral uninjured paw, labeled as (N.),before peripheral injury (t0), 17 weeks (w17) after SNI injury (n=17rats), prior to treatment (B11), SNI-paw shows increased sensitivity totouch (allodynia, decreased threshold, reflecting pain), followed by 3weekly treatments with either TCF (n=9) or Vehicle (Veh., n=8), andmonitoring touch sensitivity for 38 days, usually twice/week (3-way,repeated-measures ANOVA 3-RM-ANOVA F_(8,240)=2.33 p=0.019 forpaw*treatment*time). At week 30 (w30) a new baseline is determined and asingle treatment administered in the same groups, and behavior followedfor 38 days (3-RM-ANOVA F_(5,150)=2.22 p=0.055 for paw*treatment*time).At week 48 (w48) a third treatment trials is initiated (bl3) and tested3 days later (2-way-ANOVA for paw*treatment F_(1,28)=5.67 p=0.024),after which animals were sacrificed and brain tissue and organscollected. B. Body weight over 65 weeks, in TCF and Vehicle treated rats(no group difference). C. Open field, time in center (anxiety assessingmeasure) (no group difference). D. Open field distance traveled(mobility assessing measure) (no group difference). Red arrows=treatmentadministration. Error bars are S.E.M.s.

FIGS. 7A-7C show analysis of Purkinje neurons in long-term TCF-treatedmice by (FIG. 7A) H&E and (FIG. 7B) Nissl staining. Representativemicrographs of cerebellum from untreated (108 days) and TCF-treatedhealthy (Npc1^(+/nmf164), 225-265 days) mouse are shown. Purkinjeneurons are shown by black arrows. The images shown are representativefrom four mice in each group. (FIG. 7C) Bar diagram is quantification ofPurkinje neurons from same number of mice. Two sections per mouse wereanalyzed for counting. Numbers (mean±SD) are relative to untreatedhealthy mice. Un, untreated; Tr, treated.

FIGS. 8A-8C show safety assessment in the hippocampus of mice afterchronic treatment with TCF for long-term. (FIG. 8A) Histologicalanalysis using H&E staining. (FIG. 8B) Analysis of neuroinflammation inlong-term TCF-treated mice. Fluorescence microscopy detection ofactivated microglial cells (green, white arrows) with anti Iba-1antibodies in the hippocampus (from the regions indicated) fromuntreated and TCF-treated healthy (Npc1^(+/nmf164)). The images shownare representative from two untreated (age 108 and 109 days) and fourTCF-treated (age 225-265 days) healthy mice. (FIG. 8C) Bar diagram isquantification of microglial cells from same number of mice. Twosections per mouse were studied. Scale bar, 25 μm

FIGS. 9A-9B show assessment of chronic TCF treatment onneurobehavioral/cognitive disease score and body weight in wild typeBalb/c mice. (FIG. 9A) Major neurobehavioral symptoms and (correspondinghuman disease domain) as follows: tremor (motor); gait (ambulation);grooming (cognition); body position (cognition and motor), limb tone(motor) and weight loss (dysphagia) were assessed on a scale of 0-2except weight loss assessed as 0-3. The cumulative score is shown atindicated time points. Score of 3 or below is baseline. For 3-6 months(mo) untreated, n=15 (all males) and treated, n=17 (7 males and 10females). For 7 and 8 months untreated, n=7 (all males) and treated, n=9(4 males and 5 females), (FIG. 9B) Weight of mice in FIG. 9A. Data aremean±SD. See also FIG. 13.

FIG. 10 shows increased lung concentration of Vo in TCF injected mice.Npc1^(+/nmf164) injected i.p. with Vo or TCF. At indicated times,animals sacrificed, perfused with PBS and Vo concentration in lungs weredetermined by mass spectrometry. n=5. h, hour. *p=0.02, TCF vs Vo 0.5 h,and ** p=0.014 TCF vs Vo, 1 h, two tailed Student's t test.

FIG. 11 shows efficacy of TCF in reducing the accumulation of foamymacrophages in the lungs of Npc mice. H&E stained micrographs showingfoamy macrophages (indicated by black arrows) at 100 days of age in thelung of Npc1^(+/nmf164) (healthy control) and Npc1^(nmf164) (Npc) mutantmice treated as indicated. Foamy macrophages were abundant in untreatedNpc mice. Treatment with Vo (vorinostat) or HPBCD had no effect whereasTCF treatment greatly reduced the accumulation of foamy macrophages.Images were taken with 40× objective lens and are representative of 4mice in each group. Number of mice in each group=4. For quantitation,10-15 random fields were analyzed per lung section. Untr, Untreated.*p=0.02, TCF vs HPBCD, two-tailed Mann-Whitney test.

FIGS. 12A-12B show histological analysis of lungs from long term TCFtreated mice. Micrographs show H&E stained sections of lungs from (FIG.12A) untreated and (FIG. 12B) TCF-treated healthy (Npc1^(+/nmf164))mouse at 108 and 225 days respectively. No signs of tissue lesions,immune cell invasion or abnormal pathology were seen in long termTCF-treated mice. The images shown are representative of four untreated(108-109 days) and four TCF-treated healthy mice (age 225-265 days).Images were taken with 40× objective lens.

FIG. 13 shows assessment data for TCF-treated and untreated mice.

DETAILED DESCRIPTION

Embodiments of the present disclosure provide methods of treating andmanaging chronic pain. The methods comprise a new drug formulationcomprising a triple combination formulation (TCF). The TCF comprises,consists essentially of, or consists of, a histone deacetylase (HDAC), acyclodextrin and polyethylene glycol (PEG) or propylene glycol in atherapeutically effective amount to treat, inhibit, ameliorate, delay orreduce at least one symptom of chronic pain in a subject. In a preferredembodiment, the composition comprises, consists essentially of, orconsists of, the TCF where the HDAC may be vorinostat (Vo, a pan-HDACi),the cyclodextrin may be 2-hydroxypropyl-b-cyclodextrin (HPBCD), andpolyethylene glycol or propylene glycol may be polyethylglycol.

Histone deacetylase inhibitors (HDACi) are an important class ofemerging therapeutics approved for treating rare cancers. They elicitcomplex cellular responses by blocking HDAC enzymes. In selected geneticdisorders, HDACi induce desired transcriptional expression of genes(through histone acetylation) as well as confer indirect benefitsthrough acetylation of non-histone proteins (such as transcriptionfactors and heat shock proteins) that modulate chaperones andproteostatic networks. However, despite recognition that epigeneticmechanisms are important for brain function, treatment ofbrain-dependent diseases requires effective HDACi penetration across theblood-brain barrier (BBB). But at the same time, chronic inhibition ofHDAC function in the brain is deleterious. For example, histonedeacetylase 3 (HDAC3) is essential for Purkinje cell function andtherefore cannot be continuously antagonized, but rather HDACi dosingmust be kept low and include rest periods.

Epigenetic mechanisms and histone acetylation and deacetylation havebeen studied in animal pain models, examining peripheral, spinal cord,and cortical mechanisms. Early studies with systemically applied HDACishow confusing results. However, intrathecal injection of pan-HDACibaicalin reverses pain behavior and reduces histone acetylation in SNI.Systemically applied valproic acid and curcumin (HDACi-s) also showmodest analgesia. In an inflammatory pain model, intrathecal HDACs,vorinostat, trichostatin A (TSA), or dacinostat (LAQ824) reducedhyperalgesia and enhanced histone acetylation in the spinal cord dorsalhorn. The inventor's prior studies point to brain limbic circuitryplaying a critical role in risk for chronic pain and in adaptations thataccompany the transition into a chronic pain state. These and otherstudies imply that brain circuits involved in learning, motivation, andmood regulation all participate in the development of chronic pain andmay be targets of epigenetic changes which in turn would exert top-downregulation of nociceptive circuits. Recent evidence shows brainstemhistone H3 acetylation, and prefrontal cortex (PFC) and amygdala DNAmethylation in various animal pain models, some of which correlate withthe pain behavior. Epigenetic chronic pain-induced modifications havealso been observed in brain reward circuitry (accumbens), and HDACiinfused directly in the amygdala also reduces chronic pain-likebehavior. However, the ability to deliver HDACi across the blood brainbarrier has been limited.

This disclosure describes the use of a novel TCF that is able to crossthe blood brain barrier and to treat chronic pain. Suitable formulationsof the TCF are disclosed herein in Example 2, in Alam et al, 2017,bioRxiv (doi: doi.org/10.1101/191635)) and in Alam, M. S., Getz, M. &Haldar, K. Chronic administration of an HDAC inhibitor treats bothneurological and systemic Niemann-Pick type C disease in a mouse model.Science translational medicine 8, 326ra323,doi:10.1126/scitranslmed.aad9407 (2016), each of which are incorporatedby reference in their entirety.

For purposes of the present invention, “treating” or “treatment”describes the management and care of a subject for the purpose ofcombating the disease, condition, or disorder. Treating includes theadministration of an inhibitor of embodiments of the present disclosureto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition, ordisorder. Treating also encompasses therapeutic and palliativetreatment. The aim of treatment includes the alleviation or preventionof symptoms, slowing or stopping the progression or worsening of adisease, disorder, or condition and/or the remission of the disease,disorder or condition. The term “treat,” “treating” or “treatment” of achronic pain encompasses, but is not limited to, reducing, inhibiting,alleviating, improving, delaying or limiting at least one symptom ofchronic pain or any aspect of chronic pain. For purposes of thisdisclosure, beneficial or desired clinical results include, but are notlimited to, one or more of the following: including lessening severity,alleviation of pain and/or a symptom associated with chronic pain.

The term “effective amount” or “therapeutically effective amount” referto an amount sufficient to effect beneficial or desirable biologicaland/or clinical results. In one embodiment, the “effective amount” is anamount sufficient to reduce, inhibit, alleviate or improve one or moresymptom associated with chronic pain. An “effective treatment” refers totreatment producing a beneficial effect, e.g., amelioration of at leastone symptom of chronic pain. A beneficial effect can take the form of animprovement over baseline, i.e., an improvement over a measurement orobservation made prior to initiation of therapy according to the method.A beneficial effect can also take the form of reducing, inhibiting orpreventing at least one symptom of chronic pain, e.g. lessening theseverity of the pain, alleviating the pain or lessoning or alleviatingat least one symptom associated with chronic pain.

Reducing chronic pain and/or symptoms associated with chronic pain meansany of reducing severity (which can include reducing need for and/oramount (e.g. exposure to) other drugs and/or therapies generally usedfor pain), duration and/or frequency. Ameliorating chronic pain and/or asymptom associated with chronic pain means a lessening or improvement ofone or more symptoms of chronic pain and/or symptoms associated withchronic pain as compared to not administering the TCF. Ameliorating alsoincludes shortening or reducing a duration of a symptom.

As used herein, “delaying” the development of chronic pain means todefer, hinder, slow, retard, stabilize, and/or postpone progression ofchronic pain and/or a symptom associated with chronic pain. This delaycan be of varying lengths of time, depending on the history of thedisease and/or individuals being treated. As is evident to one skilledin the art, a sufficient or significant delay can, in effect, encompassprevention, in that the individual does not develop chronic pain. Amethod that “delays” development of the symptom is a method that reducesprobability of developing the symptom in a given time frame and/orreduces extent of the symptoms in a given time frame, when compared tonot using the method. Such comparisons are typically based on clinicalstudies, using a statistically significant number of subjects.

The term “pharmaceutically acceptable carrier” refers any carrier,diluent, or excipient that is compatible with other ingredients of theformulation and not deleterious to the recipient. A pharmaceuticallyacceptable carrier can be selected on the basis of the selected route ofadministration and standard pharmaceutical practice. The TCF may beformulated into dosage forms according to standard practices in thefield of pharmaceutical preparations. See Alphonso Gennaro, ed.,Remington's Pharmaceutical Sciences, 18th Ed., (1990) Mack PublishingCo., Easton, Pa. Suitable dosage forms may comprise, but are not limitedto, for example, solutions, parenteral solutions, injectable solutions,troches, solid preparations, suppositories, or suspensions.

In an embodiment, the TCF is preferably in unit dosage form. In suchform the preparation is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

For oral administration, the active agent may be combined with one ormore inactive ingredients for the preparation of tablets, capsules,pills, powders, granules or other suitable oral dosage forms. Forexample, the active agent may be combined with at least one excipientsuch as fillers, binders, humectants, disintegrating agents, solutionretarders, absorption accelerators, wetting agents absorbents, orlubricating agents.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, intraaural administration,intracerebral administration, rectal administration, sublingualadministration, buccal administration, and parenteral administration,including injectable such as intravenous administration, intra-arterialadministration, intramuscular administration, intradermaladministration, intrathecal administration and subcutaneousadministration. Administration can be continuous or intermittent. Invarious aspects, a preparation can be administered therapeutically; thatis, administered to treat an existing disease or condition. In apreferred embodiment, the compounds or compositions are administered byintravenous, oral, transdermal, or inhalation. In one embodiment, thecompounds or compositions are administered intraperitoneally as aninjection. In another embodiment, the compounds or compositions areadministered subcutaneously.

The terms “subject” and “patient” are used interchangeably and refer toany animal (e.g., a mammal), including, but not limited to, humans,non-human primates, rodents, and the like, which is to be the recipientof a particular treatment. Typically, the terms “subject” and “patient”are used interchangeably herein in reference to a human subject. In apreferred embodiment, the subject is a human having chronic pain.

According to a preferred embodiment of the present disclosure thechronic pain comprises one or more of chronic nociceptive pain, chronicneuropathic pain, chronic inflammatory pain, arthritis pain,fibromyalgia, breakthrough pain, persistent pain, hyperalgesia,allodynia, central sensitization, peripheral sensitization,disinhibition and augmented facilitation and cancer pain. In someembodiments, the chronic pain is cancer pain, preferably cancer painarising from malignancy or from cancer preferably selected from one ormore of: adenocarcinoma in glandular tissue, blastoma in embryonictissue of organs, carcinoma in epithelial tissue, leukemia in tissuesthat form blood cells, lymphoma in lymphatic tissue, myeloma in bonemarrow, sarcoma in connective or supportive tissue, adrenal cancer,AIDS-related lymphoma, anemia, bladder cancer, bone cancer, braincancer, breast cancer, carcinoid tumors, cervical cancer, chemotherapy,colon cancer, cytopenia, endometrial cancer, esophageal cancer, gastriccancer, head cancer, neck cancer, hepatobiliary cancer, kidney cancer,leukemia, liver cancer, lung cancer, lymphoma, Hodgkin's disease,lymphoma, non-Hodgkin's, nervous system tumors, oral cancer, ovariancancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer,stomach cancer, testicular cancer, thyroid cancer, urethral cancer, bonecancer, sarcomas cancer of the connective tissue, cancer of bone tissue,cancer of blood-forming cells, cancer of bone marrow, multiple myeloma,leukemia, primary or secondary bone cancer, tumors that metastasize tothe bone, tumors infiltrating the nerve and hollow viscus, tumors nearneural structures. Further preferably the cancer pain comprises visceralpain, preferably visceral pain which arises from pancreatic cancerand/or metastases in the abdomen. Further preferably the cancer paincomprises somatic pain, preferably somatic pain due to one or more ofbone cancer, metastasis in the bone, postsurgical pain, sarcomas cancerof the connective tissue, cancer of bone tissue, cancer of blood-formingcells of the bone marrow, multiple myeloma, leukemia, primary orsecondary bone cancer.

The composition may be administered as a single dose, or the compositioncomponents may be administered separately. For example, in someembodiments, HDAC may be administered separately from the cyclodextrinand polypropylene glycol or propylene glycol. In some embodiments, themethod includes administering HDAC before or after administering thecyclodextrin and polyethylene glycol or polypropylene glycol. In someembodiments, the method includes administering cyclodextrin beforeadministering the remaining components. In some embodiments, the methodincludes administering the HDAC separately. Preferably, however, thecomposition is administered as a single admixture.

The TCF may be administered simultaneously/concurrently or sequentially.Simultaneous administration will include the administration of thecomponents of the composition administered in different formulations,taken separately but within an hour of administration of the firstcomponent (e.g., seconds or minutes in-between). Suitably, whenadministered sequentially, for example, the HDAC may be administeredfirst followed by administration of the cycodextrin and PEG. The timebetween the administration of the different components can be adjustedfor maximum efficacy, and may be in the order of minutes or hours orlonger.

Administering may occur by different timings, for example once or morethan once. In some embodiments, the administering is carried outperiodically or substantially periodically, for example, daily, weekly,monthly, a multiple thereof, a fraction thereof, or a combinationthereof. In some embodiments, the administering is carried out daily, amultiple thereof, a fraction thereof, or a combination thereof. In someembodiments, the administering is carried out weekly, a multiplethereof, a fraction thereof, or a combination thereof. In someembodiments, the administration may occur regularly, e.g., every weekthroughout the duration of treatment, or it may occur irregularly, e.g.,once a week for a few weeks, then twice a week or not at all for a fewweeks, etc. Similarly, in some embodiments, a rest period ofnon-administration may occur between administrations. The rest periodmay occur regularly or irregularly.

In another embodiment, the disclosure provides a method of reducing orinhibition one or more symptoms of chronic pain, the method comprisingadministering to the subject a composition comprising a (i) histonedeacetylase (HDAC) inhibitor, (ii) a cyclodextrin or salt thereof, and(iii) polyethylene glycol (PEG) or propylene glycol, wherein thecomposition is provided in a therapeutically effective amount to reduceor inhibit one or more symptom of chronic pain.

The present disclosure also provides use of a composition comprising a(i) histone deacetylase (HDAC) inhibitor, (ii) a cyclodextrin or saltthereof, and (iii) polyethylene glycol (PEG) or propylene glycol formanufacture of a medicament for the treatment of chronic pain orinhibition of symptoms of chronic pain. In some embodiments, themedicament is prepared to be administered orally, sublingually, vialinhalation, transdermally, subcutaneously, intravenously,intra-arterially, intra-articulary, peri-articularly, locally orintramuscularly. In some embodiments, the HDAC inhibitor may bevorinostat, the cyclodextrin may be 2-hydroxypropyl-b-cyclodextrin(HPBCD) and the polyethylene glycol (PEG) or propylene glycol ispolyethylene glycol.

As stated above, suitable formulations of the TCF are disclosed hereinin Example 2, in Alam et al, 2017, bioRxiv (doi:doi.org/10.1101/191635), and in Alam, M. S., Getz, M. & Haldar, K.Chronic administration of an HDAC inhibitor treats both neurological andsystemic Niemann-Pick type C disease in a mouse model. Sciencetranslational medicine 8, 326ra323, doi:10.1126/scitranslmed.aad9407(2016), the contents of which are incorporated by reference in itsentirety. Suitable formulations of the TCF described therein can be usedin the methods described herein.

The dosage amount of the HDACi is an amount ranging from about 0.1 toabout 500 mg/kg. This range includes all values and subrangestherebetween, including 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350,400, 450, 500 mg/kg, or any combination thereof. In some embodiments,the dosage amount is based on a 50 mg/kg murine dose, and may be scaledfor human treatment, as is known. For example, a 50 mg/kg murine dosemay scale to 150 mg/m² in children. Such scaling is well within theskill of the artisan and may be suitably applied to any dosage for anycompound or compounds herein.

The HDAC inhibitor (HDACi) used in the present compositions include, butare not limited to, for example, hydroxamic acids, aliphatic acids,hydroxamates, benzamides, thiophene benzamide, butyrates, sodiumbutyrate, phenylbutyrate, cyclic tetrapeptide, trapoxin B, depsipeptide,cyclic peptide, electrophilic ketones, dacinostat/LAQ-824, NVP-LAQ824,givinostat/ITF-2357, bufexamac, pyroxamide, sulforaphane, trichostatin A(TSA) and analogs thereof, miglustat/OGT-918,SAHA/vorinostat/MK-0683/Zolinza, entinostat/MS-275,panobinostat/LBH-589, droxinostat/CMH, quisinostat/JNJ-26481585, PCI-24781/CRA-024781, romidepsin/FK228/FR901228/NSC 630176/depsipeptide,valproic acid, PCI-34051, CI-994/tacedinaline, M-344,rocilinostat/ACY-1215, apicidin, R-306465, mocetinostat/MGCD -0103,belinostat/PXD -101, chidamide/C S-055, abexinostat/P CI-24781, SB-939,resminostat/45C-201, kevetrin, CUDC-101, AR-42, CHR-2845, CHR-3996,4SC-202, CG-200745, ACY-1215, ME-344, RGFP-136, CBHA, AN-9, or anycombination thereof. In a preferred embodiment, the HDACi is vorinostat(Vo). In some embodiments, the use of two or more HDACi's is possible.

The cyclodextrin used in the present TCF compositions include, but arenot limited to, for example, hydroxypropyl-β-cyclodextrin,2-hydroxypropyl-β-cyclodextrin, dimethyl-β-cyclodextrin,hydroxypropyl-α-cyclodextrin, hydropropyl-γ-cyclodextrin, or anycombination thereof. In a preferred embodiment, the cyclodextrin ishydroxypropyl-β-cyclodextrin, preferably 2-hydroxypropyl-β-cyclodextrin(HPB CD). The cyclodextrin may have any average molecular weightranging, for example from about 970 to about 6,000 Da depending, forexample, on the type of cyclodextrin (α, β, or γ) and whether it iscrosslinked or uncrosslinked, substituted or unsubstituted, the degreeof substitution, and the like, as is known in the art. In someembodiments, the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin and mayhave an average molecular weight of 1396 Da. In some embodiments, two ormore cyclodextrins may be used.

In some embodiments, the cyclodextrin may be administered in an amountranging from about 1000 to about 40,000 mg/kg. This range includes allvalues and subranges therebetween, including 1000, 1200, 1400, 1600,1800, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3500, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000mg/kg, or any combination thereof. In some embodiments, the dosageamount is based on a 2000 mg/kg murine dose, and may be scaled for humantreatment, as is known.

The polyethylene glycol and propylene glycol are not particularlylimiting. In some embodiments, polyethylene glycol is used.

The molecular weight of the polyethylene glycol or polypropylene glycolis not particularly limiting. In some embodiments, the average molecularweight may range from about 100 to about 6000 Da. This range includesall values and subranges therebetween, including 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3500, 4000, 5000, 6000Da, or any combination thereof.

In some embodiments, polyethylene glycol is used, and the averagemolecular weight may range from about 100 to about 6000 Da. This rangeincludes all values and subranges there between, including 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3500, 4000,5000, 6000 Da, or any combination thereof

In some embodiments, polyethylene glycol having an average molecularweight of 100-1000 Da is used. In some embodiments, polyethylene glycolhaving an average molecular weight of 200-600 is used. In someembodiments, polyethylene glycol having an average molecular weight of400 is used. In a preferred embodiment, the polyethylene glycol 400 isused. Mixtures of polyethylene glycols having different molecularweights are possible.

The amount of polyethylene glycol is not particularly limiting. In someembodiments, the amount of polyethylene glycol may suitably range fromabout 1 to about 80% of the composition by weight. This range includesall values and subranges there between, including 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80%, orany combination thereof, based on the weight of the composition. In apreferred embodiment, PEG is used in an amount of 40-60% by weight ofthe composition, for example, 45%.

The relative amounts of HDACi:cyclodextrin:polyethylene glycol orpropylene glycol are not particularly limiting. In some embodiments, theHDACi:cyclodextrin:polyethylene glycol or propylene glycol molar ratiomay be about 1-100:1-1000:1-1000. In some embodiments, theHDACi:cyclodextrin:polyethylene glycol or propylene glycol molar ratiomay be about 1-100:1-100:1-1000. In some embodiments, the compositionhas a HDACi:polyethylene glycol molar ratio of about 1-10:1-1000:1-1000.In some embodiments, the composition has aHDACi:cyclodextrin:polyethylene glycol molar ratio of about1-10:1-100:1-1000. In some embodiments, the composition has aHDACi:cyclodextrin:polyethylene glycol molar ratio of about1:1-100:1-500. In some embodiments, the composition has aHDACi:cyclodextrin:polyethylene glycol molar ratio of about1:1-10:1-100. In some embodiments, the composition has aHDACi:cyclodextrin:polyethylene glycol molar ratio of about1:5-100:10-100. In some embodiments, the composition has aHDACi:cyclodextrin:polyethylene glycol molar ratio of about1:5-10:10-100. In some embodiments, the composition includes HDACi,cyclodextrin, and polyethylene glycol in aHDACi:cyclodextrin:polyethylene glycol molar ratio of about1-100:1-1000:1-1000. In some embodiments, the composition includesHDACi, cyclodextrin, and polyethylene glycol in aHDACi:cyclodextrin:polyethylene glycol molar ratio of about1-100:1-100:1-1000. Each of these ranges independently includes allvalues and subranges therebetween.

For example, the 1-100 range given independently includes all values andsubranges therebetween, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or anycombination thereof. Similarly, the 1-1000 range given for thecyclodextrin independently includes all values and subrangestherebetween, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, or any combination thereof. Likewise, the 1-1000 range givenindependently includes all values and subranges therebetween, including1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or any combinationthereof.

The molar ratio of HDACi:cyclodextrin is not particularly limiting, andmay suitably range from 0.001 to 100. This range includes all values andsubranges therebetween, including 0.001, 0.002, 0.003, 0.004, 0.005,0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,or any combination thereof.

Similarly, the molar ratio of HDACi:polyethylene glycol is notparticularly limiting, and may suitably range from 0.001 to 100. Thisrange includes all values and subranges therebetween, including 0.001,0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, or any combination thereof.

In some embodiments, the composition has a HDACi:cyclodextrin molarratio of less than about 0.2, ≤0.13, less than about 0.13, 0.001 to lessthan about 0.2, 0.001 to ≤0.13, 0.001 to less than about 0.13, 0.01 toabout 0.15, 0.01 to ≤0.13, 0.01 to less than about 0.13, 0.01 to ≤0.1,0.01 to less than about 0.1, 0.01 to ≤0.065, 0.01 to less than about0.065, about 0.13, or about 0.065 as appropriate.

In some embodiments, the composition has a HDACi:cyclodextrin molarratio of less than about 0.2, ≤0.13, less than about 0.13, 0.001 to lessthan about 0.2, 0.001 to ≤0.13, 0.001 to less than about 0.13, 0.01 to0.15, 0.01 to ≤0.13, 0.01 to less than about 0.13, 0.01 to ≤0.1, 0.01 toless than about 0.1, 0.01 to ≤0.065, 0.01 to less than about 0.065,about 0.13, or about 0.065 as appropriate.

In some embodiments, the composition has avorinostat:2-hydroxypropyl-β-cyclodextrin molar ratio of less than about0.2, ≤0.13, less than about 0.13, 0.001 to less than about 0.2, 0.001 to≤0.13, 0.001 to less than about 0.13, 0.01 to 0.15, 0.01 to ≤0.13, 0.01to less than about 0.13, 0.01 to ≤0.1, 0.01 to less than about 0.1, 0.01to ≤0.065, 0.01 to less than about 0.065, about 0.13, or about 0.065 asappropriate.

In some embodiments, the composition has a HDACi:polyethylene glycol orpropylene glycol molar ratio of less than 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03,0.02, 0.15, 0.01, or any combination thereof.

In some embodiments, the composition has a HDACi:polyethylene glycolmolar ratio of less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2,0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.15, 0.01,or any combination thereof.

In some embodiments, the composition has a HDACi:polyethylene glycol 400molar ratio of less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2,0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.15, 0.01,or any combination thereof.

In some embodiments, the composition has a vorinostat:polyethyleneglycol 400 molar ratio of less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02,0.15, 0.01, or any combination thereof.

The composition may or may not contain DMSO. In some embodiments, thecomposition does not contain DMSO.

In some embodiments, kits for carrying out the methods described hereinare provided. The kits provided may contain the necessary componentswith which to carry out one or more of the above-noted methods. In oneembodiment, a kit for treating chronic pain. The kit may comprise atleast one composition of the present invention and instructions for use.

It should be apparent to those skilled in the art that many additionalmodifications beside those already described are possible withoutdeparting from the inventive concepts. In interpreting this disclosure,all terms should be interpreted in the broadest possible mannerconsistent with the context. Variations of the term “comprising” shouldbe interpreted as referring to elements, components, or steps in anon-exclusive manner, so the referenced elements, components, or stepsmay be combined with other elements, components, or steps that are notexpressly referenced. Embodiments referenced as “comprising” certainelements are also contemplated as “consisting essentially of” and“consisting of” those elements. The term “consisting essentially of” and“consisting of” should be interpreted in line with the MPEP and relevantFederal Circuit's interpretation. The transitional phrase “consistingessentially of” limits the scope of a claim to the specified materialsor steps “and those that do not materially affect the basic and novelcharacteristic(s)” of the claimed invention. “Consisting of” is a closedterm that excludes any element, step or ingredient not specified in theclaim.

The present disclosure has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of thedisclosure.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting examples.

EXAMPLES Example 1 TCF Use in Treatment of Chronic Pain

A new drug formulation comprising a triple combination formulation (TCF)comprising Vo (a pan-HDACi), the caging agent2-hydroxypropyl-b-cyclodextrin (HPBCD), and polyethylene glycol (PEG)(FIG. 1) was formulated as a therapeutic for neurodegenerativedisorders, and shows efficacy in a mouse model of Niemann-Pick type Cdisease. TCF boosts the ability of HDACi to cross the blood brainbarrier. In the mouse, intraperitoneal (ip) TCF increases histoneacetylation in the brain, and low-dose repeated TCF treatment inNpc1^(nmf164) −/−mice increases Npc1 transcript and protein levels inthe brain, and extends animal survival by 100% (See Alam et al, 2017,bioRxiv (doi: doi.org/10.1101/191635, incorporated by reference).Importantly, even after 8-12 months of once-weekly ip TCF, normal miceshowed no detectable toxicity (See Alam, M. S., Getz, M. & Haldar, K.Chronic administration of an HDAC inhibitor treats both neurological andsystemic Niemann-Pick type C disease in a mouse model. Sciencetranslational medicine 8, 326ra323, doi:10.1126/scitranslmed.aad9407(2016), incorporated by reference in its entirety.

The Example shows that ip TCF in rats with spared nerve injury (SNI; avalidated rodent model for human chronic pain) decreases the primaryneuropathic pain-related behavior, tactile allodynia, by 50% for aduration of about 30 days. This analgesia seems quite specific as TCFdid not interfere with tactile sensitivity of the uninjured paw, andpain relief was not accompanied with motor or anxiety-like deficits. Thesustained long-duration and large magnitude of pain relief with TCF isitself very exciting as no other clinically available painpharmacotherapy shows such properties.

Our data in the SNI rat and one-year TCF-treated mice demonstrate theydo not exhibit obvious signs of toxicity.

Medicinal chemistry approaches have attempted to develop brain permeantand selective HDACi (and KDACi). But the path of new chemical entitiesto the clinic is long (and to date none have yet been chronicallyadministered). In contrast the TCF boosts brain penetration of a FDAapproved Vo delivered with GRAS (generally regarded as safe) compoundsHPBCD and PEG, which are also well tolerated. While brain concentrationsof Vo achieved confer substantial neurological benefit in mice, they arelow (which could indeed explain the observed tolerance). For multipleyears, the inventors have been testing various chemical formulations aspotential therapies for chronic pain. The ability of this novel TCFformulation rendering the HDACi vorinostat (Vo) to cross the BBB butwith no detectable metabolic toxicity has been surprisingly found to beable to also control chronic neuropathic pain. This Example demonstratespain relief and for providing a tool with exciting potential to exploresustained central epigenetic control of chronic pain.

Simultaneously increasing BBB permeation to functional CNS-active levelsand repeated administration of Vo via the TCF without toxicity achieveslong awaited milestones in the utilization of an HDACi in the treatmentof brain epigenetically modulated disease conditions, such as chronicpain.

The theoretical design and proposed action of the TCF is shown inFIG. 1. In mice, TCF boosted Vo levels in plasma and in brain by 2-3fold (FIGS. 2A-2B). The final maximal levels of Vo in whole brainremained low (˜1 ng/mg), but these levels were sufficient to stimulatehistone acetylation (FIGS. 3A-3B) and increase Npcl transcript andprotein levels in the brain as well as Npc1^(nmf164) mouse survival (seeFIGS. 6B and 6C and FIGS. 2C-E in Alam et al.¹). Vo in PEG provided nobenefit. 2× HPBCD provided no additional benefit compared to 1× HPBCDalone, revealing that Vo in TCF directly impacted survival. Notably,even after 8-12 months of once-weekly ip administration in normal micethe TCF has shown no toxicity in liver or kidney function, does notinduce weight loss or long-term behavioral dysfunction. In brains ofthese animals, there is no loss of Purkinje neurons in the cerebellum orinflammation in hippocampus (FIGS. 4 and 5A-5B).

To test for TCF efficacy in chronic pain we used the spared nerve injury(SNI) model⁴⁹ in Sprague-Dawley adult/aging male rats. SNI rats showpain-like behavior, tactile allodynia and cold hyperalgesia, for therest of life post-SNI. The model has been used extensively, and we andothers show mechanistic equivalences for brain adaptations betweenhumans with chronic pain and SNI rats or mice^(3,10,17,19,20,50-53).

Unilaterally SNI-injured rats when treated with TCF, TCF(SNI) vs.Veh.(SNI) (once/week, 3 treatments), showed robust and sustainedanalgesia (about 50%) that outlasted the treatment by >20 days. Thetreatment effect is specific as the uninjured paw sensitivity was notaffected, TCF(N.) vs. Veh.(N.). When the procedure was repeated with asingle treatment, specific analgesia was sustained for >30 days. A thirdtreatment session (monitoring behavior for 3 days) replicated thespecific analgesia observed (FIG. 6A). Body weight did not differbetween TCF (n=9) or Vehicle (n=8) treated SNI animals over the 50 weeks(FIG. 6B). Also, anxiety and mobility were not different between the twogroups (FIGS. 6C and 6D). TCF treated SNI rats groomed normally and hadhealthy appearing fur. The long-lasting, relatively large analgesia,with no obvious signs of non-specific effects or off-target toxicity,makes this formulation an exciting candidate to explore for epigeneticcontrol of chronic pain.

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Example 2

Histone deacetylase inhibitors (HDACi) are emerging therapeutics for abroad range of diseases including cancer and neurodegeneration¹⁻⁴. Theyblock HDAC enzymes, to promote acetylation of both histones andnon-histone proteins to elicit complex cellular changes^(5,6).HDACi-induced histone modifications have been shown to increase ordecrease transcriptional expression of mutated target gene(s) in manygenetic diseases as well as indirect benefit through modulation ofchaperone and proteostatic networks ⁷-9. Due to their broad effects ontranscription, it is particularly important to maximize HDACi efficacywhile limiting dose. We previously reported on development andvalidation of a therapeutic strategy of a triple combination formulation(TCF) of the HDACi vorinostat (Vo) that enabled lowering concentrationsof Vo to treat cerebral disease as well as inflammation in liver andspleen, in a mouse model of a fatal cerebellar disorder Niemann-PickType C (NPC) disease N.

NPC is caused by defect in either Npc1 or Npc2 genes¹¹. It is a rareautosomal recessive neurodegenerative disease. 95% of cases are due todefect in Npc1. Cells with defects in either Npc genes accumulatecholesterol late endosomes/lysosomes^(12,13). A point mutation in Npc1gene that blocks cholesterol transport in cells is causative forneurodegeneration in a mouse model¹⁴. At the organismal level, in thecentral nervous system (CNS), Npc1 is essential for myelination¹⁵ andlikely additional functions¹⁶. Neurodegeneration is a hallmark ofclinical NPC disease. Disease progression can be heterogeneous and slowbut once initiated, is invariably fatal¹¹. Splenomegaly and hepatomegalyare common presenting symptoms in pediatric cases followed byneurocognitive and neuromuscular degeneration¹⁷. Lung disease isprominent and can even be cause of death^(18,19).

Presently the only available treatment for NPC is miglustat (Zavesca™),an iminosugar that decreases glycosphingolipid accumulation in type1Gaucher's disease^(20,21), was approved for NPC treatment in Europe,Canada and Japan but denied FDA approval (although it is prescribed offlabel in the US). Miglustat may confer mild improvement in specificclinical symptoms but fails to prevent disease progression^(22,23).2-Hydroxy propyl beta cyclodextrin (HPBCD) is being investigated as anemerging therapy^(24,25.) It chelates cholesterol but does not cross theblood brain barrier²⁶. Therefore, to treat neurological disease HPBCDmust be directly delivered to the central nervous system (CNS;^(27,28))which carries procedural risk of life-long therapy. Systemic delivery isneeded to improve liver and other visceral organs but inexplicably,HPBCD is excluded from lung^(29,30) and therefore of little benefit toend-stage advanced and frequently fatal bronchial disease. Arimoclomolis another emergent therapy for NPC³¹, but its benefit for systemicdisease especially in treatment of lung inflammatory disease remainsunknown.

The TCF combines HPBCD, PEG, and Vo in a defined formulation¹⁰. Uponsystemic injection, it increases the plasma exposure of Vo and boostsits delivery across the BBB to stimulate histone acetylation there.Although mice chronically treated for close to a year showed nometabolic toxicity¹⁰, the effect of long term TCF exposure on keyneurons, brain areas and overall progression of symptoms ofneurodegeneration that mimic human disease, have not been assessed.Further, while HPBCD reduces systemic inflammation^(10,24,29) it isexcluded from lungs^(29,30) and therefore whether the TCF promotes Vodelivery and therapeutic action in lungs remains unknown. Our findingson these points advance development of a new HDACi therapeutic strategyto treat NPC and other difficult-to-treat disorders that may benefitfrom epigenetic therapy.

Methods

Materials—All fine chemicals including 2-hydroxypropyl-β-cyclodextrin(HPBCD) and polyethylene glycol 400 (PEG) were procured from Sigma (StLouis, Mo., USA) unless otherwise indicated. Vorinostat was from SelleckChemicals (Houston, Tex., USA).

Animals—Npc1^(nmf164) is a BALB/c strain carry a D1005G (A to G at cDNAbp 3163) mutation in the Npc1 gene 32. A breeding pair of mutant micewere obtained from Robert P. Erickson, University of Arizona HealthSciences Center, Tucson, Ariz., USA and is available at The JacksonLaboratories. Homozygous mutants (Npc1^(nmf164)) along with wild-typelittermates (Npc1^(+/+)), were generated in house by crossingheterozygous mutant (Npc1^(+/nmf164)) males and females and genotyped aspreviously described¹⁰. Wild type Balb/c mice were procured from Envigo(Indianapolis, Ind., USA).

Drug injection and organ harvest—The Triple combination formulation(TCF) is a mixture of vorinostat (50 mg/kg), HPBCD (2000 mg/Kg)), PEG400 (45%) and DMSO (5%). Vorinostat (50 mg/Kg) was made in 5% DMSO and45% PEG. HPBCD was a 20% (w/v) solution and given dose of 2000 mg/Kg.Detailed methodology on preparing drug solutions have been descriedearlier¹⁰. To enable comparative studies with prior regiments, all micewere given two doses of HPBCD at P7 and P15. From P21 onwards, micereceived either HPBCD alone or TCF, as indicated. Vo was also initiatedat P21. Injections were administered weekly through the intraperitoneal(i.p) route (and the injection volume used was 10 ml/Kg body weightacross all treatment groups). For lung histopathology, Npc1^(nmf164)mice were analyzed at 100-109 days of age. Long-term safety was assessedfor 8-10 months either in Npc1^(+/nmf164) or commercially purchased wildtype Balb/c mice. The animals were sacrificed by asphyxiation using CO2and harvested organs were immersed fixed in 10% neutral bufferedformalin (˜4% formaldehyde) for 24 hours at RT and subsequently storedin 70% alcohol until transfer to paraffin.

Nissl and H&E staining—Paraffin-embedded sections (4-5 μm) were dewaxedin xylene and alcohol. For Nissl, brain sections were stained withacidified 0.1% cresyl violet for 7 min followed by two incubations in95% ethanol of 5 min each. The sections were cleared in xylene andmounted in cytoseal XYL (Thermo Scientific, Kalamazoo, USA). H&Estaining of brain and lung tissues was carried out by AML laboratoriesaccording to standard methods³³. Images were visualized with DPIan Apo40×/1.00 oil immersion objective lens (Nikon) and captured on a NikonOlympus microscope, using a Nikon digital DS-Fi1-U2 camera controlled byNIS-Elements F3.0 Nikon software (all from Nikon Instruments INC, Tokyo,Japan).

Iba1 immunostaining of brain sections—Paraffin-embedded brain sections(4-5 μm) were dewaxed in xylene and alcohol. Iba1 antigen was retrievedby boiling the sections in acidic condition for 30 min. Blocking wasdone with 2% goat serum for 30 min at RT. Sections were incubated withanti-Iba1 (1:500, 019-19741, Wako Chemicals) overnight at 4° C.FITC-conjugated secondary IgG antibodies (MP Biomedicals, Solon, Ohio,USA) were used at a dilution of 1:200. Nuclei were stained with DAPI(0.5 m/ml) and mounting was done using Vectashield (Vectorlaboratories). Sections were visualized with 40× oil-immersion objectivelens (NA 1.35) and image collection was performed using an Olympus IXinverted fluorescence microscope and a Photometrix cooled CCD camera(CH350/LCCD) driven by DeltaVision software from Applied Precision(Seattle, Wash., USA). DeltaVision software (softWoRx) was used todeconvolve these images. Images are single optical sections. Images wereanalyzed using ‘softWoRx’ or ‘ImageJ’ software (NIH, MD, USA).

Quantification of Vo in lungs—Npc1^(+/nmf164) mice (age 6-7 weeks) weregiven intraperitoneal injections of either Vo (50 mg/Kg in 45% PEG and5% DMSO) or TCF (Vo 50 mg/Kg+HPBCD, 2000 mg/Kg+PEG, 45%+DMSO, 5%). At 30min and 1 h post injection, mice were asphyxiated with CO2, blood wasdrawn by cardiac puncture and organs were perfused with 20 ml ice-coldPBS through the ventricle. Harvested lungs were cut into small pieces(4-6 mm2) and flash frozen in liquid nitrogen. The quantification ofvorinostat was done by Metabolite Profiling Facility, Bindley BioscienceCenter, Purdue University, IN, USA. The detailed methods are asdescribed earlier¹⁰. Briefly, the tissue was homogenized using aPrecelly bead homogenizer system utilizing ceramic CK 14 beads. 2 ng ofdeuterated internal standard (d5-Vorinostat, Toronto Research Chemicals,Ontario, Canada) was added to lung homogenate prior to liquid extractionwith acetonitrile. Prior to analysis, samples were reconstituted in 100μL of 50% water/50% acetonitrile. An Agilent 1200 Rapid Resolutionliquid chromatography (HPLC) system coupled to an Agilent 6460 seriestriple quadrupole mass spectrometer (MS/MS) was used to analyzevorinostat. The data were obtained in positive electrospray ionization(ESI) mode and quantitated by monitoring the following transitions: forVorinostat, 265→232 with a collision energy of 5 V and ford5-Vorinostat, 270→237 with collision energy of 5 V.

Results

Assessment of chronic TCF-treatment in cerebellar and hippocampalregions as well as a neurobehavioral/cognitive disease score in mice.HDACs are important enzymes and their functions are required in braindevelopment³⁴⁻³⁶, in particular, HDAC3 knockdown blocked development ofPurkinje neurons³⁷. It has therefore been hypothesized that long-termHDACi treatment may adversely affect the brain. However, we havereported that weekly administration of the TCF in Npc1^(nmf164) miceprevented loss of Purkinje cell neurons¹⁰. Since Vorinostat from the TCFpeaks at 30 minutes and is rapidly cleared from the brain and plasma¹⁰,our findings suggested that epigenetic modulation associated withtransient block of HDAC3 (as well as other HDACs) may be tolerated andbenefit NPC-diseased animals.

But the effects of TCF on neurons in normal animal brain remainunaddressed. Since Purkinje are major neurons requiring HDAC function,we used them as a sentinel neuron for effects of extended TCF-treatmentin healthy animals. We administered weekly TCF to heterozygous, healthy‘control’ animals for 2-3 fold longer (240-300 days) than the 100day-efficacy period in Npc1^(nmf164) mice. As shown in FIG. 7A, H&Estaining failed to show any change in histological features of Purkinjecells in the cerebellum. Nissl staining suggested they were intactneurons (FIG. 7B). Quantitative analyses of both H&E—and Nissl—stainingconfirmed that the TCF even on extended treatment did not lead to deathand loss of Purkinje cells (FIG. 7C). These data indicate thatrecurrent, short-lived exposure of low concentrations of vorinostat doesnot cause neuronal loss, even as it is sufficient to trigger sustainedepigenetic effects.

Activation of microglial cells marks neuroinflammation, an early sign ofneuronal dysfunction^(38,39). We have previously shown that microgliastained with antibodies to the Iba inflammatory marker accumulate in thehippocampus of Npc1^(nmf164) mice¹⁰. Further at ˜100 days after weeklyTCF treatment, Iba staining is reduced suggesting that TCF can targetinflammatory dysfunction in the hippocampus. Comparable H&E staining isseen in representative hippocampal regions from mice exposed to weeklyTCF treatment for 225-265 days compared to untreated animals at 100-110days (FIG. 8A) with each showing only a few resident microglial cells(FIG. 8B). Quantitative analysis showed no significant difference in thenumber of microglial cells untreated and chronically TCF-treated mice(FIG. 8C), suggesting that despite the fact that Vo is predicted totranscriptionally activate numerous target genes, the TCF does notinduce an inflammatory response broadly damaging to neurons.

Clinically NPC disease is defined by major and minor symptomaticdomains, whose severity has been quantified to monitor the naturalhistory of the disease⁴⁰. While awaiting plasma biomarkers⁴¹⁻⁴⁴ symptomscoring continues as an important index of progressive disease. Wepreviously created a disease severity scale for murine NPC that capturesmajor patient disease domain scored in a defined indicated range andwhose sum provides the cumulate disease score, (with a maximal score of13;¹⁰). Because older healthy animals, particularly males, oftendisplayed poor grooming and slight impairment in limb tone onwards of100 days, a cumulative score of 3 or higher reliably flags onset ofsymptomatic disease. Untreated Npc1^(nmh164) mice progress to a score of10-13 by 100 days, but TCF affords significant reduction to 4-5 whenadministered over the same period¹⁰ to render functional benefit tomajor symptomatic domains of neurological disease that includeambulation, cognition, motor control and dysphagia. In contrast, thecumulative score remained below baseline in healthy wild type micereceiving chronic weekly administration of TCF (FIG. 9A, FIG. 13; withindicated scores of 1-2 that also appear in untreated animals, asexpected due to their poor grooming). There was also no change in animalweight (FIG. 9B) showing TCF-treatment did not impair overall nutrientconsumption and utilization (which marks mid- and end-stage neurologicaldisease¹⁰).

TCF increases Vo levels in lung. As previously reported the TCF is atriple combination formulation containing Vo, HPBCD, and PEG¹⁰. In priorwork we found that 1 h after injection of TCF, Vo concentrations inmouse plasma were 3 fold higher compared to the levels observed when Vowas administered in PEG alone¹⁰. Vo levels in the brain were alsosignificantly boosted in TCF-injected mice¹⁰. These data suggested thatthe HPBCD was a major contributor to the pharmacokinetic (PK) effect inplasma and brain. Further examination of brain, liver, and spleensuggested the TCF could treat both neurological as well as systemic NPCdisease in mice. However, since HPBCD is known to be excluded fromlungs^(29,30), it remained unclear whether the TCF increased exposure ofVo and/or benefit lung disease.

As shown in FIG. 10, animals injected with Vo in PEG alone showed a meanconcentration of 3.2 ng/mg Vo in lungs at 30 minutes, which decreased to1 ng/mg by 60 min. After TCF injection, Vo concentration reached 7.9ng/mg at 30 min and then declined to 4.2 ng/mg at 60 min. These datasuggested that the TCF boosted Vo entry into lungs, likely due to the(2.5-3 fold) plasma pharmacokinetic effect (previously reported in¹⁰).Vo concentrations (of 4.2 ng/mg) detected at 60 min in TCF-treatedanimals were reduced by 45-50% reduction from levels seen at 30 min.Animals injected with Vo in PEG showed a 65-70% reduction over the sameperiod, suggesting that in addition to boosting peak concentrations, theTCF may also slow down Vo clearance from lungs and both effects mayincrease levels and exposure of Vo in lungs.

TCF reduces the accumulation of foamy macrophages in the lungs ofNpc1^(nmf164) mice. Previous studies^(24,29,45) have shown that thesystemic delivery of HPBCD in NPC mice fails to alleviate lung disease(because HPBCD may not be able to reach the tissue). Since HPBCD is amajor component of the TCF, this raised question on whether theformulation could alleviate lung disease even as it boosted Vo deliveryto other organs. To test this, we undertook histochemical analysis oflungs from control and treated mice. Animals were examined at 100 daysof age, since in prior work with the Npc1^(nmf164) mouse model we haveshown that this is a time of significant neurological disease responsiveto treatment by TCF. As shown in FIG. 11, H&E stained micrographsrevealed accumulation of large number of foamy macrophages in the lungsof untreated Npc1^(nmf164) mice at 100 days. Semi-quantitative analysisshowed TCF treatment significantly reduced the number of macrophages(FIG. 11). In contrast, administration of Vo alone or HPBCD continued tobe associated with abundant macrophage accumulation (FIG. 11). Togetherthese findings suggest that TCF-induced increase of Vo in lungs andreduced inflammation there.

Long-term chronic treatment with TCF shows no deleterious effect on lunghistopathology in healthy control animals. Since our treatment analysesin Npc1^(nmf164) were undertaken at 100 days, the effects of extendedTCF administration in control Npc1^(+/nmf164) type animals were assessedat 2-3 times longer periods (200-300 days). We previously reported thatanalysis of metabolic markers in the plasma failed to reveal toxicity inthe liver and kidneys of mice treated once weekly with TCF after 200-300days¹⁰. Histological features of liver were also found to be normal¹⁰.In FIGS. 12A-12B, we show that lungs of Npc1^(+/nmf164) mice at 200-300days show absence of tissue lesions, immune cell invasion or anyabnormal pathology, as determined by H&E staining.

Discussion

Concerns about intrinsic toxicity of HDACi are pertinent for both panHDACi and inhibitors designed to target a given HDAC, since even asingle HDAC can regulate hundreds of genes (and hence the value ofsynthesizing selective HDACi has been debated). Since neurologicaltreatments may be long term, it is important to learn the effects ofextended treatment periods well beyond when efficacy is detectable,especially in the brain. Our data in FIGS. 7A-7C, 8A-8C, and 9A-9Bsuggest that the TCF enables chronic administration of a therapeuticallyviable dose of broad spectrum HDACi with no detectable histologicalchanges in key brain regions and neurocognitive/behavioral functions inmice. Purkinje neurons are major neurons that participate in motorcontrol and learning. They can both emit and receive signals andfunction to regulate the entire cerebellum. Thus, maintenance ofPurkinje cells provides a single read out for complex neuronal processin the cerebellum but also communication from the spinal cord and brainstem. Our data showing that the TCF helps preserve Purkinje cells in theNPC disease model, suggests these cells are responsive to HDACi (likelydue to elevation of NPC1 protein but possibly also by indirectmechanisms). Therefore, our finding that extended exposure to weekly TCFfor 8 to 10 months had no effect on Purkinje neuron staining or count,suggests HDACi administration via the TCF is well tolerated inPurkinje-associated as well as overall cerebellar functions. Similarly,the hippocampus located in the cerebrum and a key region for learningand memory, shows no adverse structural and inflammatory effects despiteextended TCF exposure. Although assessment of neurocognitive andbehavioral scores do not yield quantitative tissue analyses, theyindicate that TCF does not induce symptoms (and therefore processes) ofneurodegeneration in wild type mice, even though it can delay appearanceof these diseased processes in the NPC mouse model. Finally, findingsthat the TCF can boost delivery of vorinostat into lungs and reducerecruitment of macrophages into alveolar spaces, suggests that althoughHPBCD is excluded, vorinostat released from the TCF gains access lungslikely due the plasma exposure. Vo levels delivered to lungs are boostedto sufficiently reduce macrophage levels in Npc1^(nmf164) mice, whichsignificantly expands the potential of the TCF in treating all organsystems expected to affect the progression of NPC. Extended TCFadministration showed no ill-effects on lung pathology of normal mice.

To conclude, extended TCF administration failed to induce adverseeffects on metabolic parameters, brain, and neurological functions aswell as visceral organs including lung, although the TCF shows efficacyin all of these domains in the NPC mouse model. This may appear to becounterintuitive, since Vo is a broad acting HDACi at thetranscriptional level with potential to target thousands of genes.However, proteomics studies suggest that changes may be limited to ˜200targets in NPC diseased cells⁴⁶. Moreover, control healthy cells do notshow major changes in proteome readouts in response to Vo⁴⁶. Oneexplanation for this difference may be that mechanisms that restorenormalcy in diseased cells are distinct from those that maintainhomeostasis in normal cells.

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Each publication, patent, and patent publication cited in thisdisclosure is incorporated in reference herein in its entirety. Thepresent invention is not intended to be limited to the foregoingexamples, but encompasses all such modifications and variations as comewithin the scope of the appended claims.

1. A method of treating chronic pain in a subject in need thereof, themethod comprising administering to the subject a composition comprising(i) a histone deacetylase (HDAC) inhibitor, (ii) a cyclodextrin or saltthereof, and (iii) polyethylene glycol (PEG) or propylene glycol,wherein the composition is provided in a therapeutically effectiveamount to treat the chronic pain.
 2. The method of claim 1, wherein theHDAC inhibitor is vorinostat.
 3. The method of claim 1, wherein thecyclodextrin is 2-hydroxypropyl-b-cyclodextrin (HPBCD).
 4. The method ofclaim 1, wherein the polyethylene glycol (PEG) or propylene glycol ispolyethylene glycol.
 5. The method of claim 1, wherein the compositioncomprises a molar ratio of HDAC inhibitor:cyclodextrin:PEG of about1-100:1-1000:1-1000.
 6. The method of claim 1, wherein the HDACinhibitor is present in an administration amount of about 0.1-500 mg/kg,cyclodextrin is present in an administration amount of about 1000-40,000mg/kg, and the PEG is present in an amount of about 30-60% compositionby weight.
 7. The method of claim 1, wherein the HDAC inhibitor isvorinostat at an administration amount of about 50 mg/kg, thecyclodextrin is HPBCD at an administration amount of at about 2000mg/kg, and PEG is about 40-50% composition by weight.
 8. The method ofclaim 1, wherein the chronic pain comprises one or more of, chronicnociceptive pain, chronic neuropathic pain, chronic inflammatory pain,arthritis pain, fibromyalgia, breakthrough pain, persistent pain,hyperalgesia, allodynia, central sensitization, peripheralsensitization, disinhibition and augmented facilitation, or cancer pain.9. A method of reducing or inhibiting one or more symptoms of chronicpain, the method comprising administering to the subject a compositioncomprising (i) a histone deacetylase (HDAC) inhibitor, (ii) acyclodextrin or salt thereof, and (iii) polyethylene glycol (PEG) orpropylene glycol, wherein the composition is provided in atherapeutically effective amount to reduce or inhibit one or moresymptom of chronic pain.
 10. The method of claim 9, wherein the HDACinhibitor is vorinostat.
 11. The method of claim 9, wherein thecyclodextrin is 2-hydroxypropyl-b-cyclodextrin (HPBCD).
 12. The methodof claim 9, wherein the polyethylene glycol (PEG) or propylene glycol ispolyethylene glycol.
 13. The method of claim 9, wherein the compositioncomprises a molar ratio of HDAC inhibitor:cyclodextrin:PEG of about1-100:1-1000:1-1000.
 14. The method of claim 9, wherein the HDACinhibitor is present in an administration amount of about 0.1-500 mg/kg,cyclodextrin is present in an administration amount of about 1000-40,000mg/kg, and the PEG is present in an amount of about 30-60% compositionby weight.
 15. The method of claim 9, wherein the HDAC inhibitor isvorinostat at an administration amount of about 50 mg/kg, thecyclodextrin is HPBCD at an administration amount of at about 2000mg/kg, and PEG is about 40-50% composition by weight.
 16. The method ofany one of claim 9, wherein the chronic pain comprises one or more of,chronic nociceptive pain, chronic neuropathic pain, chronic inflammatorypain, fibromyalgia, breakthrough pain, persistent pain, hyperalgesia,allodynia, central sensitization, peripheral sensitization,disinhibition and augmented facilitation, or cancer pain.
 17. Use of acomposition comprising (i) a histone deacetylase (HDAC) inhibitor, (ii)a cyclodextrin or salt thereof, and (iii) polyethylene glycol (PEG) orpropylene glycol for manufacture of a medicament for the treatment ofchronic pain or inhibition of symptoms of chronic pain.
 18. The useaccording to claim 17, wherein the medicament is prepared to beadministered orally, sublingually, vial inhalation, transdermal,subcutaneously, intravenously, intraperitoneally, intra-arterially,intra-articulary, peri-articularly, locally, or intramuscularly.
 19. Theuse according to claim 18, wherein the medicament is prepared to beorally or intravenously administered.
 20. The use according to claim 17,wherein the HDAC inhibitor is vorinostat, the cyclodextrin is2-hydroxypropyl-b-cyclodextrin (HPBCD) and polyethylene glycol (PEG) orpropylene glycol is polyethylene glycol.
 21. The use according to claim17, wherein the composition comprises a molar ratio of HDACinhibitor:cyclodextrin:PEG of about 1-100:1-1000:1-1000.
 22. The useaccording to claim 17, wherein the HDAC inhibitor is present anadministration amount of about 0.1-500 mg/kg, cyclodextrin is present inan administration amount of about 1000-40,000 mg/kg, and the PEG ispresent in an amount of about 30-60% composition by weight.
 23. The useaccording to claim 17, wherein the HDAC inhibitor is vorinostat at anadministration amount of about 50 mg/kg, the cyclodextrin is HPBCD at anadministration amount of at about 2000 mg/kg, and PEG is about 40-50%composition by weight.
 24. The use according to claim 17, wherein thechronic pain comprises one or more of, chronic nociceptive pain, chronicneuropathic pain, chronic inflammatory pain, fibromyalgia, breakthroughpain, persistent pain, hyperalgesia, allodynia, central sensitization,peripheral sensitization, disinhibition and augmented facilitation, orcancer pain.